The goal of this proposal is to test the novel hypothesis that disruption of synchrony between brain-gut circadian rhythms either directly by alcohol, or by environmental or genetic manipulations, is the vulnerability factor responsible for the differential susceptibility for EtOH-induced intestinal hyperpermeability that explains why only subset of alcoholics develop gut leakiness to endotoxins and steatohepatitis [ASH]. Our hypothesis is supported by: (1) Gut leakiness is a major contributor to endotoxemia and gut-derived endotoxin is required for ASH; (2) while EtOH universally disrupts intestinal epithelial monolayer permeability, gut leakiness occurs in only a subset of alcoholics, suggesting other factors might be involved-variability in gut leakiness; (3) The core circadian clock molecular machinery is within all organs including the central circadian clock in the hypothalamic suprachiasmatic nucleus (SCN), and intestinal epithelial cells. The SCN regulates and coordinates the expression and timing of multiple peripheral circadian molecular rhythms, possibly including brain-gut interactions of the so called brain-gut axis, (BGA); (4) The BGA can regulate intestinal permeability, and pathological stimuli like physical and psychological stress can cause gut leakiness; (5) The circadian modulation of the brain gut communication could affect intestinal permeability since circadian genes regulate apical junctional complex (AJC) protein genes that are directly involved in regulation of intestinal permeability. Our recent in vivo mice data showed that disruption of circadian rhythms makes the intestine susceptible to injury. Also, our pilot data in Caco-2 intestinal monolayers show that alcohol stimulates expression of the clock genes Clock and Per2 and that siRNA knockdown of these genes prevents alcohol-induced monolayer hyperpermeability. We also show that Clock and Per2 proteins are increased in the intestines of alcohol fed rats with leaky gut. To test our hypothesis, we will take two different approaches. First (in Aim 1), we will use both environmental [constant phase shifts in the entraining LD cycle] & genetic [Clock mutant and Per1/Per2 KO mice] approaches to disrupt the overall circadian organization of mice to determine if such disruption leads to increased vulnerability for EtOH-induced gut leakage to endotoxins in alcohol-fed mice (8 wk chronic model). We predict that these circadian manipulations will elucidate the roles of circadian brain-gut synchrony and intestinal cell clock genes in regulating intestinal apical tight junctional proteins and gut permeability in response to chronic alcohol feeding. Second (in Aim 2), we will assess how EtOH-induced changes in central and peripheral clock function and gene expression impacts permeability to endotoxin (3 day acute and 8 wk chronic models). We predict that alcohol-mediated central and/or intestinal circadian desynchrony will result in increased injury to apical junctional complex (AJC) leading to gut leakiness. Demonstrating that circadian- mediated disrupted brain-gut communication is one critical contributing susceptibility factor for alcohol- induced endotoxemia would provide new targets for preventive and therapeutic interventions in ASH.